Direct effects of arsenic trioxide on action potentials in isolated cardiac tissues: importance of the choice of species, type of cardiac tissue and perfusion time.

INTRODUCTION: The aim of the present study was to evaluate direct/acute effects of arsenic trioxide on action potentials (APs) in isolated cardiac tissues, and to investigate if the choice of species and tissue and the duration of the perfusion play a role in arsenic-induced acute/direct prolongation of AP/QT. METHODS AND RESULTS: Direct electrophysiological effects of arsenic trioxide were measured in cardiac tissues isolated from four different species using micro-electrode recording. Arsenic (after 30 to 95 min perfusion at 10 µM) significantly prolonged APD(90), increased triangulation of the AP and elicited early afterdepolarizations (EADs) only in isolated guinea-pig and dog Purkinje fibers but not in rabbit and porcine (minipig) Purkinje fibers. Arsenic induced a prolongation of the APD(90) and increases in triangulation and the occurrence of EADs was not observed in papillary muscles of guinea-pigs and rabbits. Arsenic at 4 increasing concentrations from 0.1 µM to 10 µM at the standard perfusion-time of 15 min per concentration, and after a continuous 90-min perfusion at 1 µM and 1 Hz did not induce these direct effects on APD(90), triangulation and EADs in isolated guinea-pig Purkinje fibers, but it at 1 µM elicited EADs in 2 out of 7 preparations after 90 min at 0.2 Hz. DISCUSSION: The present study demonstrates that the choice of species and cardiac tissue as well as perfusion-time play important roles in arsenic-induced direct/acute effects on APD(90) and induction of EADs in vitro.

Predicting drug-induced slowing of conduction and pro-arrhythmia: identifying the 'bad' sodium current blockers.

BACKGROUND AND PURPOSE: The regulatory guidelines (ICHS7B) for the identification of only drug-induced long QT and pro-arrhythmias have certain limitations. EXPERIMENTAL APPROACH: Conduction time (CT) was measured in isolated Purkinje fibres, left ventricular perfused wedges and perfused hearts from rabbits, and sodium current was measured in Chinese hamster ovary cells, transfected with Na(v)1.5 channels. KEY RESULTS: A total of 355 compounds were screened for their effects on CT: 32% of these compounds slowed conduction, 65% had no effect and 3% accelerated conduction. Lidocaine and flecainide, which slow conduction, were tested in more detail as reference compounds. In isolated Purkinje fibres, flecainide largely slowed conduction and markedly increased triangulation, while lidocaine slightly slowed conduction and did not produce significant triangulation. Also in isolated left ventricular wedge preparations, flecainide largely slowed conduction in a rate-dependent manner, and elicited ventricular tachycardia (VT). Lidocaine slightly slowed conduction, reduced Tp-Te and did not induce VT. Similarly in isolated hearts, flecainide markedly slowed conduction, increased Tp-Te and elicited VT or ventricular fibrillation (VF). The slowing of conduction and induction of VT/VF with flecainide was much more evident in a condition of ischaemia/reperfusion. Lidocaine abolished ischaemia/reperfusion-induced VT/VF. Flecainide blocked sodium current (I(Na)) preferentially in the activated state (i.e. open channel) with slow binding and dissociation rates in a use-dependent manner, and lidocaine weakly blocked I(Na). CONCLUSION AND IMPLICATIONS: Slowing conduction by blocking I(Na) could be potentially pro-arrhythmic. It is possible to differentiate between compounds with 'good' (lidocaine-like) and 'bad' (flecainide-like) I(Na) blocking activities in these models.

Choice of cardiac tissue in vitro plays an important role in assessing the risk of drug-induced cardiac arrhythmias in human: beyond QT prolongation.

INTRODUCTION: Recently we have demonstrated that to the choice of tissue type is important in identifying I(Kr) and I(Ks)-induced prolongation of the action potential. However, the differential sensitivity of cardiac tissues to other ionic current blockers or modulators is relatively unknown. The aim of the present study was therefore to evaluate tissue-specific effects of different ion channel blockers or activators on the action potential (AP), which can affect other parameters in addition to drug-induced APD/QT prolongation or shortening. METHODS AND RESULTS: Electrophysiological effects were measured in isolated rabbit Purkinje fibers, papillary muscles and ventricular trabeculae using a microelectrode technique under the following conditions: block of I(to) with 4-AP (1 x 10(-3) M), block of Ca(2+) channels with diltiazem (1 x 10(-5) M), block of Na(+) channels with flecainide (1 x 10(-5) M), activation of Ca(2+) current with Bay-K-8644 (1 x 10(-5) M), activation of K(ATP) channels with levcromakalim (1 x 10(-5) M) or block of I(K1) current with BaCl(2) (n=8 to 12 for each group). 4-AP prolonged APD significantly more in the Purkinje fiber than in the papillary muscle or the ventricular trabecula. 4-AP elicited 63% incidence of early afterdepolarizations but 0% in the papillary or trabeculae. Diltiazem and flecainide shortened APD(40) and APD(50) and increased triangulation more in the Purkinje fiber, whilst having little effect on these parameters in the papillary muscle or the ventricular trabecula. Bay-K-8644 significantly prolonged APD in the ventricular trabecula, but not in the Purkinje fiber or the papillary muscle. BaCl(2) prolonged APD(90) in all tissues, but significantly shortened APD(40) only in the Purkinje fiber. Levcromakalim shortened APD in all tissues, but significantly less in the Purkinje fibers. CONCLUSION: The present study demonstrates that certain cardiac tissues respond differently to the same ion channel blockers/activators, which are not involved in APD/QT prolongation. As such the appropriate selection of tissue needs to be taken into careful consideration in cardiac safety assessments when exploring different mechanisms of drug-induced changes in the action potential.

Calmodulin antagonist W-7 prevents sparfloxacin-induced early afterdepolarizations (EADs) in isolated rabbit purkinje fibers: importance of beat-to-beat instability of the repolarization.

INTRODUCTION: The occurrence of early afterdepolarizations (EADs) has been related to the incidence of torsades de pointes in drug-induced long QT (LQT). The generation of EADs may be facilitated by Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase). METHODS AND RESULTS: In the present study, we investigated a possible involvement of Ca(2+)/Calmodulin dependent protein kinase in the generation of sparfloxacin-induced EADs in isolated rabbit Purkinje fibers by means of a calmodulin antagonist W-7. EADs were evident in 8 of the 10 preparations perfused with sparfloxacin at 1 x 10(-4) M and stimulated at 0.2 Hz. The induction of EADs by sparfloxacin was associated with a large prolongation of the duration of the action potential (APD), an increase in the triangulation, and the short-term instability of the repolarization. CaM kinase blockade with the calmodulin antagonist W-7 inhibited sparfloxacin-induced EADs in a concentration-dependent manner (EADs were induced in 3 of 10, 1 of 10, and 0 of 8 preparations in the presence of W-7 at 5 x 10(-7) M, 5 x 10(-6) M, and 5 x 10(-5) M, respectively; P < 0.01 at 5 x 10(-6) M and 5 x 10(-5) M). The inhibition of sparfloxacin-induced EADs by W-7 at 5 x 10(-7) M and 5 x 10(-6) M was associated with a significant decrease in the beat-to-beat instability but not associated with a significant shortening of the APD and reduction of V(max). CONCLUSION: The present findings support the hypothesis that CaM kinase may be a proarrhythmic signaling molecule and demonstrate that CaM kinase may be involved in the generation of EADs in drug-induced LQT and enhanced beat-to-beat instability of repolarization is essential for the genesis of EADs in rabbit in vitro.

Choice of cardiac tissue plays an important role in the evaluation of drug-induced prolongation of the QT interval in vitro in rabbit.

INTRODUCTION: Regulatory guidelines (CPMP/986/96, ICHS7B) recommend the use of isolated cardiac tissues, including Purkinje fibers, papillary muscles and ventricular trabeculae, for detecting potential drug-induced long QT. However, the differential sensitivity of these tissues in experimental drug-induced long QT is relatively unknown. We investigated the electrophysiological characteristics of these tissue types in vitro together with their different responses to drugs that are known to induce prolongation of the QT interval in man. METHODS: Electrophysiological parameters were measured in vitro using a micro-electrode technique. The isolated rabbit Purkinje fibers, papillary muscles or ventricular trabeculae were superperfused with Tyrode's solution and stimulated according to different stimulation protocols. The effects of dofetilide (1 x 10(-8) M), sertindole (1 x 10(-6) M), erythromycin (3 x 10(-4) M) and sparfloxacin (1 x 10(-4) M) were evaluated relative to solvent (n=8 to 12 for each group). RESULTS: In isolated Purkinje fibers, action potential duration at 90% repolarization (APD(90) at 1 Hz) was markedly prolonged by 55% (erythromycin), 103% (dofetilide), 118% (sertindole) and 88% (sparfloxacin) from baseline. The prolongation of APD(90) caused by these 4 compounds was associated with a 28% to 78% incidence of early afterdepolarizations (EADs) at 0.2 Hz only in the Purkinje fiber. In contrast, APD(90) was altered by erythromycin, dofetilide, sertindole and sparfloxacin only by +15%, +6%, -7% or +15%, respectively, in isolated papillary muscles, and by 33%, +28%, +4% or +16%, respectively, in ventricular trabeculae. EADs were not induced by these four compounds in papillary muscles or in trabeculae. Reducing the stimulation rate to 0.2 Hz resulted in a 33% prolongation of APD(90) in Purkinje fibers, while APD(90) was shortened by 10% in papillary muscles and by 20% in ventricular trabeculae. CONCLUSION: The present study demonstrates that the differential sensitivity of tissue types play an important role in the detection of drug-induced long APD and EADs. Indeed the Purkinje fiber was the only tissue type to display the well known phenomenon associated with I(kr) channel blockade (inverse-use dependence), when the stimulation rate was decreased below 1 Hz. Rabbit Purkinje fibers constitute the most sensitive tissue type for detecting drug-induced long action potential duration and EADs. As such the selection of tissue type needs to be taken into careful consideration in cardiac safety assessments when exploring drug induced long QT.